W.F. Bayne

Determination of imipenem (N-formimidoyl thienamycin) in human plasma and urine by high-performance liquid chromatography, comparison with microbiological methodology and stability

High-performance liquid chromatographic (HPLC) methods using ultraviolet (UV) detection have been developed for the assay of the antibiotic imipenem (N-formimidoyl thienamycin) in human plasma and urine. A reversed-phase analytical column is employed in the plasma assay method and a cation-exchange column is used in the urine assay method. Both methods use borate buffer in the mobile phase. The method of preparation of human fluid samples for HPLC injection has been optimized with respect to the stability of imipenem in aqueous buffers, in morpholine buffer--ethylene glycol stabilizer, and in urine and plasma. Preparation of the samples before injection into the HPLC systems involves deproteination/filtration of the plasma/urine samples. The open lactam metabolite and the coadministered dehydropeptidase inhibitor, cilastatin sodium, do not interfere with the 313-nm detection of imipenem in either the plasma or the urine assay. Thienamycin, the precursor of imipenem and an impurity in imipenem formulations, is separated from the drug using both of these methods. Concentrations generated from the HPLC analysis of plasma and urine samples from two healthy volunteers compare favorably with results using a microbiological assay method. Correlation of the two methods gives r greater than or equal to 0.990 for both fluids.

Determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid in urine by automated pre-column derivatization with 2,3-naphthalene dicarboxyaldehyde and high-performance liquid chromatography with fluorescence detection

A sensitive (5 ng/ml) method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid in human urine is described. The procedure includes (1) the isolation of the drug from urine by co-precipitation of its calcium salt with endogenous phosphates in the presence of base, (2) a solid-phase anion-exchange sample clean-up and (3) automated pre-column derivatization of the primary amino group with 2,3-naphthalene dicarboxyaldehyde-cyanide reagent followed by fluorescence detection of the N-substituted cyanobenz[f]isoindole derivative. The derivative of the drug was synthesized and its spectral and fluorescence properties were evaluated. The fluorescence quantum efficiency was determined to be 0.82 in the mobile phase used for the assay. The derivative is also capable of accepting energy in an oxalate ester-hydrogen peroxide chemiluminescence system.

Simultaneous high-performance liquid chromatographic analysis of carbidopa, levodopa and 3-O-methyldopa in plasma and carbidopa, levodopa and dopamine in urine using electrochemical detection

Two assay procedures are described for the analysis of levodopa, carbidopa and 3-O-methyldopa in plasma and levodopa, carbidopa and dopamine in urine. The methods are suitable for quantifying the analytes following therapeutic administration of levodopa and carbidopa. Both were based on reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection and with methyldopa as the internal standard. Plasma samples were prepared by perchloric acid precipitation followed by the direct injection of the supernatant. Urine was prepared by alumina adsorption, and the analytes were desorbed with perchloric acid solution containing disodium EDTA and sodium metabisulfite prior to injection into the HPLC system. The methods have been utilized to evaluate the pharmacokinetics and bioavailability of oral dosage forms containing levodopa and carbidopa.

Comparison of Plasma Profiles of Lovastatin (Mevinolin), Simvastatin (Epistatin) and Pravastatin (Eptastatin) in the Dog

SummaryLovastatin (mevinolin) [LV], simvastatin (epistatin) [SV] and pravastatin (eptastatin) [PVA] are indicated in the treatment of hypercholesterolaemia and are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Beagle dogs were dosed orally with LV and SV (both lactones), PVA (a hydroxy acid), LVA (the β-hydroxy acid of LV) and SVA (the β-hydroxy acid of SV), in separate experiments. Measurement of enzyme inhibitory activities showed that the plasma concentrations were considerably higher after dosing with the hydroxy acids LVA, SVA and PVA than after the lactones LV and SV, for equivalent doses. Plasma samples were also assayed for active metabolites by a specific high performance liquid chromatography (HPLC) method; these were LVA and SVA, together with their 6′-β-hydroxy, 6′-β-hydroxymethyl, and 6′-exomethylene analogues. These metabolites were present in roughly equal concentrations after dosing with the lactones LV and SV. The unchanged drug was the major component after dosing with the hydroxy acids LVA, SVA and PVA. The major metabolite present after dosing with LVA and SVA was the 6′-exomethylene analogue. This work demonstrates a basic difference in plasma concentrations and metabolic profiles between the lactones LV and SV and the hydroxy acids LVA, SVA and PVA after oral administration to the dog.

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of a novel 4-aza-steroidal inhibitor of 5 alpha-reductase in human plasma has been developed. The assay is based on a single solid-phase extraction and an efficient HPLC separation on two analytical columns in series. The assay has been fully validated and used to support Phase II and III clinical pharmacokinetic studies. The lowest limit of quantification was found to be at 1 ng/ml and allowed pharmacokinetic evaluation of the drug at doses down to 5 mg.

Simultaneous quantification of cycloserine and its prodrug acetylacetonylcycloserine in plasma and urine by high-performance liquid chromatography using ultraviolet absorbance and fluorescence after post-column derivatization.

D-Cycloserine is a broad-spectrum antibiotic used with other antibiotics to treat various forms of tuberculosis. Its prodrug sodium (R)-4-[(1-methyl-3-oxo-1-butenyl)amino]-3-isoxazolidinone hemihydrate, developed for better aqueous stability and solubility, is combined with another broad-spectrum antibiotic, fludalanine. An ion-pair, reversed-phase high-performance liquid chromatographic assay has been developed to simultaneously detect cycloserine and its prodrug in plasma and urine. The prodrug is detected directly by ultraviolet absorbance and cycloserine by fluorescence following post-column derivatization.

High-performance liquid chromatographic determination of cilastatin in biological fluids

Cilastatin, a dehydropeptidase-I inhibitor, is coadministered with the beta-lactam antibiotic imipenem. The described procedure was developed for quantification of cilastatin in human plasma and urine. The assay involved sample purification on a C18 extraction cartridge, reversed-phase high-performance liquid chromatography with post-column derivatization and fluorescence detection. Standard curves were linear from 0.75 to 75.0 micrograms/ml in plasma and from 2.5 to 200.0 micrograms/ml in urine. Intra-day mean coefficients of variation at concentrations within the standard curve range were 4.2 +/- 2.4% and 3.1 +/- 1.7% in plasma and urine, respectively. The inter-day coefficients of variation for analyses of cilastatin in plasma (1.0 and 50.5 micrograms/ml) were less than 10% after 31 days of analysis while those for urine (5.0 and 74.1 micrograms/ml) were less than 11% after 44 days of analysis. The limits of reliable detection were 0.75 and 2.5 micrograms/ml in plasma and urine, respectively. This procedure met the sensitivity and specificity requirements for the analysis of samples from clinical pharmacokinetic studies.

Analysis of sulindac and metabolites in plasma and urine by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with ultraviolet detection is described for the quantification of sulindac, sulindac sulfone and sulindac sulfide in plasma and sulindac, trans-sulindac, sulindac sulfone and sulindac sulfide in urine. Plasma samples are de-proteinized with acetonitrile and urine samples are injected directly following enzymatic hydrolysis of glucuronide metabolites. The resulting chromatograms are essentially free from endogenous interference and the limits of detection are 0.1 microgram/ml for plasma and 0.2 microgram/ml for urine for all of the above compounds.

High-performance liquid chromatographic method for post-column, in-line derivatization with o-phthalaldehyde and fluorometric detection of phenylpropanolamine in human urine

A rapid and sensitive method for the analysis of phenylpropanolamine in urine was developed using high-pressure liquid chromatography, post-column, in-line derivatization with o-phthalaldehyde, followed by detection with a fluorometer. Human urine was injected directly into the chromatographic system, and phenylpropanolamine separated in the reversed-phase mode. The practical lower limit of detection was 0.1 microgram/ml of urine. The coefficient of variation from 0.96 to 96 micrograms/ml varied between 2.23 and 0.19%, respectively. The linearity of the calibration graphs is excellent (r = 0.9999) over a concentration range of two orders of magnitude.

Determination of the antibiotic fludalanine in plasma and urine by high-performance liquid chromatography using a packed-bed, post-column reactor with o-phthalaldehyde and 2-mercaptoethanol

Fludalanine is a novel anti-bacterial agent active against gram-negative and gram-positive bacteria. A high-performance liquid chromatographic assay has been developed using ion-pair chromatography to resolve fludalanine and the internal standard 3,3-difluoro-D-alanine from plasma and urine background. The mobile phase contains sodium dodecyl sulfonate and methanol in a phosphate buffer. Fludalanine is derivatized post-column with o-phthalaldehyde via a packed-bed chemical reactor. The adduct is detected fluorometrically. The plasma and urine assays are sensitive to 0.25 and 0.5 micrograms/ml, respectively.